(a) Field of the Invention
The present invention relates to a method for genetic detection and a primer set for a polymorphic marker site. More particularly, the present invention relates to a primer set for a polymorphic marker site having a primer encompassing unique information, and to a method for genetic detection using the primer set.
(b) Description of the Related Art
Genomes ceased to be viewed as static entities when mobile interspersed genetic elements were first discovered in maize in the 1940's. Interspersed elements are DNA segments, which retain the ability to move their conserved sequence information from one genomic region to another via some enzyme-facilitated process. Since their discovery they have been found to reside ubiquitously throughout different genomes. They vary in frequency from one species to another and in most cases their function has remained questionable. Only recently has DNA sequencing technology shed light on the impact mobile genetic elements have on the genome in which they reside. The human genome project revealed a mobile element composition of approximately 45%.
There are several types of interspersed genetic elements in the human genome: Long INterspersed Elements (LINEs), Short INterspersed Elements (SINEs), SVA elements (other SINE, VNTR and Alu combination), and Human Endogenous Retroviruses (HERVs) to name a few. LINEs are a major retrotransposon group as they make up approximately 21% of the mass of the human genome. LINEs can be 4-6 kilobases in length and facilitate their own movement by producing mobilization enzymes. SINEs are elements approximately 300 base-pairs in length and produce no proteins with which to facilitate their movement. SINEs have, however, been extremely successful in the human genome, constituting more than 10% of the mass of the human genome.
Interspersed genetic elements have increased our understanding of the formation of human populations as well as non-human populations through the field of molecular evolution. Molecular evolution studies have identified human genetic diversity within and between continental populations. The fundamental basis for human identity testing is that genetic markers vary as to the presence/absence between individuals in the population. This is called polymorphism. Interspersed genetic elements are known for their contribution to human genetic variation because of their polymorphism. Recently, there have been large-scale, genome-wide studies geared to locating polymorphic elements. Because of these studies, advances have been made in the field of human population genetics. Geographic affiliation research now exists which utilizes these elements and is designed to identify the geographic origin of an unknown DNA sample. These polymorphic elements now have an entire public database devoted to their mapped chromosomal location.
There are several characteristics, which make interspersed genetic elements appealing to use as human identity testing markers and other genetic testing markers, and have an advantage over traditionally used Short Tandem Repeats (STRs). There is a distinct subfamily structure and this hierarchy is associated to how recently the Alu elements inserted into the genome. The “younger” elements are polymorphic in human populations. They are identical by descent markers: individuals that share an element do so because they share a common ancestor. They are stable inserts because once an element integrates there is no known excision mechanism with which to remove it from a genomic position. The possibility of another genetic element landing in the same genomic spot is essentially zero. Also, the ancestral state of an element is also known to be effectively the absence of that element. Currently used STRs are subject to mutation through the cells own DNA copying mechanisms and thus, can promote doubt in the mind of the tested participants.
Two previous publications (Novick et al., International Pediatrics 9: 60-68 (1994); Novick et al., Electrophoresis 16: 1596-1601 (1995)) utilizing Alu interspersed genetic elements for paternity and human identity testing was introduced, however, these studies were exclusive to using Alu elements because they were short enough in sequence, were not multiplex reactions, but single biological reactions, and used a different primer design methodology which will ultimately cause false results.
Interspersed genetic elements have the most potential to cause genomic deleterious effects because they are still actively mobilizing. They can cause insertion, recombination (swapping genetic material between sequences which are similar) mutations and transduce genetic material from one chromosomal location to another. Insertion mutation can occur when the element integrates into a promoter region, coding region or non-coding region. Alteration or complete halt of gene expression has been attributed to a few insertions. Homologous recombination events can occur between elements which are in close genomic proximity to one another. Non-homologous recombination between elements has also been shown. It has been suggested that insertion and recombination mutagenesis caused by Alu elements accounts for approximately 0.4% of human genetic diseases.
Creating multiplex PCR reactions using interspersed genetic elements up till now has been problematic due to several reasons. These elements encompass roughly 45% of the entire mass of the human genome. This essentially means that the same sequence from a particular type of element is found millions of times scattered randomly throughout all human chromosomes. Because of this homology (sequence similarity) there is difficulty in targeting a single polymorphic marker site (one that varies from individual to individual) because the polymorphic marker sites are the same sequence as the fixed (a marker site that does not vary between individuals). Another difficulty is getting uniqueness from a single polymorphic marker site.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.